Figure 5. CD4⁺ T_FH cells are expanded post-COVID-19.

a, b. Unsupervised clustering of CD4⁺ T cells from adenoids and tonsils according to flow cytometric surface markers (a). Quantification of the effect of prior SARS-CoV-2 infection on CD4⁺ T cell clusters showing regression coefficients ± 95% confidence intervals (CI) and p values, estimated with a linear model controlling for age and sex (b). Significantly different clusters (p<0.05) between post-COV and UC groups are indicated with a star or highlighted in red. Adenoids: post-COV n=12, UC n=38; tonsils: post-COV n=15, UC n=43. c, d. Frequencies of manually-gated CD45RA⁺CCR7⁺ naïve CD4⁺ T cells (p=0.022 for tonsils) (c) and CD57⁺PD-1^hi CD4⁺ T cells (p=0.001 for adenoid) (d) in post-COV and UC adenoids and tonsils (adenoids post-COV n=17, UC n=42; tonsils post-COV n=18, UC n=46). e. Plots of CD69 and CXCR5 expression on CD57⁺PD-1^hi CD4⁺ T cells and total CD4⁺ T cells from one tonsil, representative of tonsils and adenoids from 26 donors. f. Image of post-COV adenoid showing CD57⁺PD-1^hi CD4⁺ T cells in one GC, representative of tonsils and adenoids from 6 donors. Magnification of square inset shown on the right. CD4 in cyan, CD57 in yellow, and PD-1 in magenta. GC boundaries defined using Ki-67 (see Figure 1h). 1:CD4⁺CD57⁺, 2:CD4⁺PD-1⁺, 3:CD4⁺CD57⁺PD-1⁺ cells. Scale bars 100μm (left) and 10μm (right). g. Cytokine combinations (IFN-γ, IL-2, IL-10, IL-17A, IL-21 and TNF, as analyzed by SPICE) produced by tonsillar or adenoid CD4⁺ T cells from post-COV (n=13) and UC (n=13) donors following PMA and ionomycin stimulation (category 27: p=0.04, 33: p=0.01, 41: p=0.03). h,i. Unsupervised clustering of CD4⁺ T cells from PBMC (h) and quantification of the effect of prior SARS-CoV-2 infection (i) as described in a, b (post-COV n=13, UC n=34). j. Frequencies of CD45RA⁻CXCR5⁺PD-1⁺ circulating T_FH (cT_FH) and CXCR3⁺CCR6⁻ cT_FH cells in post-COV (n=16) and UC (n=41) PBMCs, p=0.032 for CXCR3⁺CCR6⁻ cT_FH cells. Sample list for panels a-d and h-j in Supplementary Table 2 and for panel g in Supplementary Table 11. Each symbol represents one donor. Means ± S.D. displayed in bar plots. Significance calculated with two-sided Mann-Whitney U test. *p<0.05, **p<0.01.